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BOR - Papers in Press, published online ahead of print October 27, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.032623
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BIOLOGY OF REPRODUCTION 72, 502–508 (2005)
DOI: 10.1095/biolreprod.104.032623
© 2005 by the Society for the Study of Reproduction, Inc.

Identification of a New, Testis-Specific Sperm Antigen Localized on the Principal Piece of the Spermatozoa Tail in the Fox (Vulpes vulpes)1

Yann Verdier3,4, Guillaume Farré3, Nelly Rouet3, Zoltan Kele4, Tamás Janáky4, and Franck Boué2,3

Agence Française de Sécurité Sanitaire des Aliments,3 Laboratoire d'études et de recherches sur la rage et la pathologie des animaux sauvages, Unit of Wildlife Health and Management, F-54220 Malzéville, France Department of Medical Chemistry,4 University of Szeged, H-6720 Szeged, Hungary


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fox (Vulpes vulpes) sperm antigens were identified to assess them as a potential target for a contraceptive vaccine. We report here the cloning and sequencing of fSP13, a fox sperm protein of 97 kDa. The fSP13 protein was both auto- and iso-antigenic in foxes; it was recognized by sera of foxes immunized with fox sperm proteins and vasectomized foxes. The NH2-terminal sequence of fSP13 was determined, and a piece of cDNA was amplified from testicular RNA by reverse transcription polymerase chain reaction. This piece was used to screen a cDNA library from fox testis by Southern blot. A sequence of 1662 base pairs was obtained, including a major open reading frame coding for 498 amino acid. Mass spectrometry analysis confirmed the position of the open reading frame and the presence of posttranscriptional modifications. Analysis of the predicted amino acid sequence revealed no apparent transmembrane regions. Comparison of the protein sequence with the Prosite database demonstrated the presence of four potential N-linked glycosylation sites. The fSP13 bears the closest amino acid similarity to two human sperm proteins: fibrousheathin 2 and testis-specific calcium binding protein 86-VII. The deduced 80 N-terminal amino acid sequence also presents similarity with the RII{alpha} domain. By using a serum against fSP13, this antigen was localized on the principal piece of the fox spermatozoa. Northern blot analysis showed that fSP13 is specifically expressed in testis. The fSP13 is one of the first fox sperm antigens to be cloned and sequenced.

fox, gamete biology, sperm, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In various species, the persistence of serum antisperm antibodies has been associated with infertility in experimental models of immunization with isologous or autologous sperm antigens [1], following reversed vasectomy [2], and in unexplained cases of both male and female infertility [3]. These antibodies are considered to be induced by sperm antigens. Many of those are tissue-specific proteins, which are synthesized and incorporated into spermatozoa only at puberty, long after the neonatal period during which the immune system sorts self from nonself [4]. Production of antibodies is usually avoided because, by puberty, the contact between sperm antigens and the immune system in the male is restricted because male germ cells are sequestered behind the blood-testis barrier during the last stages of their development [5].

Identification of sperm antigens would be useful for the identification of novel sperm-specific proteins, for understanding the formation of antisperm antibodies and development of tools for diagnosis of infertility and immunocontraceptive vaccines. Because of the interspecies differences in the biochemical composition of the sperm, this identification must be done individually for each species. In the case of the fox (Vulpes vulpes), only a few sperm proteins have been identified and biochemically characterized: FSA-1 [6], LDH-C4 [7], and PH-20 [8]. It is important to identify more fox sperm proteins, in particular, to provide potential target proteins for a contraceptive vaccine [9].

The immunological approach has been used to identify sperm proteins [10]. In a previous study on fox sperm antigens, Verdier et al. [11] showed that sera containing antisperm antibodies are obtained after immunization of foxes and rabbits with fox sperm surface proteins. These sera were used to localize seven highly antigenic proteins on two-dimensional (2D) gel electrophoresis of fox sperm proteins.

Similarly, antisperm antibodies have been detected in sera of vasectomized foxes [12]. Vasectomy usually induces an immunologic response, such as cell-mediated antisperm immunity and a rise of antisperm auto antibodies [13–15]. While the frequency of the appearance of antisperm antibodies is known in various species, only a few sperm autoantigens, such as protamine [16] and FA-1 [2], have been precisely identified by using sera from vasectomized animals. Using 2D gel electrophoresis of fox sperm proteins and Western blotting, eight areas containing major antigens were localized [12]. Some proteins are immunoreactive with the sera from these two studies [11, 12], indicating a high antigenic potential.

The objective of this study was the molecular characterization of a high-molecular weight (MW) auto- and iso-antigen of fox spermatozoa, recognized by sera from immunized rabbits and foxes and vasectomized foxes. This antigen, named fSP13, has a MW of 97 kDa and an isoelectric point (i.p.) of 4.3–4.6.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals

All chemicals were provided by Sigma, St. Louis, MO, except when it is specifically stated otherwise.

Preparation of Spermatozoa, Extraction of Sperm Proteins, and Purification of fSP13 Using 2D Electrophoresis

Sperm samples were obtained from 50 wild foxes (V. vulpes), which had been caught for rabies monitoring during the reproduction period in the Northeast of France (January–March). Epididymides were dissected and the postmortem sperm was collected from the cauda epididymis by retrograde flushing from the vas deferens toward the proximal cauda with phosphate buffer saline (PBS: 140 mM NaCl, 16 mM KCl, 6.7 mM Na2HPO4, 1.4 mM KH2PO4, 1 mM MgCl2; pH 7.3). Spermatozoa samples were pooled, washed twice with PBS by centrifugation (5 min at 800 x g at room temperature) and pellets of 108 spermatozoa were stored at –80°C until analyzed (maximum 9 mo). For the 2D electrophoresis, each pellet was solubilized in 200 µl of a solution of 9.5 M urea, 2% Igepal, 2% 3–10 Ampholines (Amersham Bioscience, Orsay, France) and 5% ß-mercaptoethanol in bidistillated water.

The 2D gel electrophoresis was based on the procedure of O'Farrell [17]. Sperm proteins were run on a first dimensional 4% urea gel pre-equilibrated with 9.5 M urea and pH 3–10 ampholines. The evaluation of the i.p. as a function of the migration distance was carried out as described previously [11]. The second dimension electrophoresis was performed in a 12% acrylamide gel at 12°C under reducing conditions according to [18]. Gels were then stained with Coomassie blue according to [19]. Spots of fSP13 were carefully cored from the gel and used for Western blotting, microsequencing, and mass spectrometry.

For the identification of fSP13 as an antigen, 10 spots of the purified protein were pooled and tested by Western blot with sera collected from immunized rabbits and foxes and from vasectomized foxes, as previously described [11, 12]. Briefly, protein was blotted onto a nitrocellulose membrane. The blot was saturated 1 h with 5% nonfat milk (Biorad, Marnes la Coquette, France) in PBS, washed with PBS, and incubated overnight in presence of the fox or rabbit sera, diluted 1:1000 in PBS. Then the membrane was washed three times with PBS-Tween. In the case of the fox sera, the membrane was incubated for 1 h with a rabbit anti-dog IgG diluted 1:1000 (Nordig, Tilburg, Netherlands) and washed three times with PBS-Tween. The membrane were then incubated for 1 h with peroxidase-conjugated anti-rabbit IgG at 1:30 000 (Sigma) and washed with PBS-Tween. The immune complexes were detected with chemo-luminescent substrate of peroxidase (Pierce, Rockford, IL) according to the supplier's instructions.

Microsequencing of fSP13

Twenty spots of the protein fSP13 electroblotted onto polyvinyldenedifloride membrane (PVDF) (Millipore, Bedford, MA) were stained with Coomassie blue R-250 and the relevant band excised and subjected to N-terminal amino acid (AA) sequence analysis [20] on an Applied Biosystem gas phase sequencer (Model 476) equipped with a phenylthiodantoin analyzer (Model 120A). The sequencing program was run as recommended by the manufacturer. Amino acid sequences obtained were then used to scan databases (NCBI and EMBL) for similarity to known proteins.

Generation of a fSP13 cDNA Probe

For testis collection, two fertile foxes were obtained from the Norwegian Fur Breeders Association (Eidsvoll, Norway). They were housed individually in outdoor cages in compliance with the Canadian Council on Animal Care guidelines in our experimental farm (agreement number A 54747). Testes were frozen during reproduction season and stored at –80°C. Tissue was homogenized in liquid nitrogen, then total RNA was extracted using Trizol (Invitrogen Life Technologies, Renfrewshire, Scotland) and mRNA was purified with Oligotex kit (Qiagen, Courtaboeuf, France) according to the manufacturer's instruction.

Reverse transcription was performed using moloney murine leukemia virus reverse transcriptase and oligodT primers (Applera, Paris, France). The polymerase chain reaction (PCR) was performed using hot start Taq platinum DNA polymerase (Invitrogen Life Technologies) with forward primer (5'-TGCTTATGATCA AGCTCCTGAG-3') and reverse primer (5'-CCTTCTGCATCCAGGAGTTG-3') designated from the sequence of the fibrousheathin 2 (FSP2) homologue and synthesized by Genome Express (Paris, France). Thermal cycling was done in mastercycler gradient thermal cycler (Eppendorf, Hambourg, Germany) using a program of one cycle at 94°C for 5 min, 35 cycles of 94°C for 1 min, 56°C for 30 sec, and 72°C for 1 min, and a last cycle at 72°C for 10 min. PCR products were separated by electrophoresis on 2% Agarose gel stained with ethidium bromide. A 369-base pair (bp) DNA fragment was extracted from the gel with Genelute column (Sigma) and cloned in pUC18/SmaI vector (Amersham Bioscience) according to the manufacturer's instruction. Plasmids were purified with Perfectprep plasmid mini kit (Eppendorf), screened and sequenced on a Perkin-Elmer Biosytem DNA sequencer using Dye terminator Chemistry (ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit; Applied Biosystem, Courteboeuf, France) with M13 forward primer (5'-AGC GGATAACAATTTCACACAGG-3') and M13 reverse primer (5'-CCCAGTCACGACGTT GTAAAACG-3'). Purified plasmids from positive clones were digested with the restriction enzymes XbaI and PstI.

Construction and Screening of Fox Testis cDNA Library

Fox testis cDNA library was realized using ZAP Express cDNA synthesis kit and ZAP Express cDNA gigapack III gold cloning kit (Stratagene, Amsterdam, Netherlands), according to the manufacturer's instruction. After phages plating and growing, they were blotted in duplicate onto a nitrocellulose (+) membrane (Amersham Biosciences). Membranes were treated 2 min in 0.2 M NaOH, 1.5 M NaCl; 5 min in 0.5 M Tris-HCl (pH 8), 1.5 M NaCl; and 1 min in 0.2 M Tris-HCl (pH 7.5), saline-socium citrate (SSC) 2x; and DNA was fixed by cross linking by ultraviolet irradiations. The library was screened by Southern blot.

Labeling of DNA Probe with digoxigenin and detection were realized using DIG High prime DNA labeling and detection starter kit II (Roche diagnostics, Meylan, France) according to the manufacturer's instruction. Hybridization was performed 16 h at 42°C. Then, 1.5 x 105 phages were plated for the first screening. Positive clones were purified by isolation of the plaque and two additional screening procedures. After the last screening, positive clones were converted into plasmids using helper phage as described in the manufacturer's protocol. Plasmids pBK-CMV containing the target insert were sequenced by gene walking, as described. The first primers used were T3 forward (5'-AATTAACCCTCACTAAAGGG-3') for sense sequence and T7 reverse (5'-GTAATACGACTCACTATAGGGC-3') for antisense sequence. The other primers (forward and reverse) were synthesized to cover all the sequences.

Mass Spectrometry

Spots of protein fSP13 were excised from 10 Coomassie blue-stained 2D electrophoresis gels and analyzed by mass spectrometry [21]. The gel plugs were diced into small pieces and destained by washing twice with water and acetonitrile. Protein was reduced in gel with 10 mM dithiothreitol in 100 mM NH4HCO3 for 30 min at 56°C and alkylated with 55 mM iodoacetamine in 100 mM NH4HCO3 for 45 min in the dark at room temperature. The alkylation solution was removed, and the gel pieces were washed once with 100 mM NH4HCO3, dehydrated with acetonitrile, and dried in a vacuum centrifuge. The gel pieces were swollen in digestion buffer containing 50 mM NH4HCO3, 5 mM CaCl2, and 12.5 ng/µl trypsin for 30 min on ice. The digestion buffer was exchanged with 50 mM NH4HCO3/5 mM CaCl2 and samples were digested overnight at 37°C. Peptides were extracted from gel pieces in 5% (v/v) formic acid for 1 h, followed by three extractions with 50:50 acetonitrile/5% (v/v) formic acid for 1 h. The combined extracts were reduced to 20 µl in a vacuum centrifuge and the peptides were purified on a C18 ZipTip (Millipore Corporation) according to the manufacturer's instructions. Samples were mixed at a 1:1 ratio with a saturated aqueous solution of 2,5-dihydroxybenzoic acid matrix.

Mass spectrometric analyses were carried out on a Bruker Reflex IV MALDI time-of-flight mass spectrometer (Bruker-Daltonics, Bremen, Germany) equipped with a nitrogen laser (337 nm). Spectra were collected in positive ion reflector mode with delayer extraction. Ion acceleration voltage was 20 kV. The spectra were calibrated externally using peptide standards, but if necessary, they were internally recalibrated on trypsin autodigestion products (m/z 842.50 and 2211.10). The monoisotopic masses for all peptide ion signals in the acquired spectra were determined and used for comparison with theoretic spectra of the fSP13 cDNA sequence.

Generation of Polyclonal Serum Against fSP13 and Immunofluorescence Localization on Fox Sperm

The peptide used for raising the antibody is derived from the hydrophilic region of fSP13 (EKWSEGTTQEKEPEC) [22]. The peptide was coupled to keyhole limpet lymphocyanin as carrier according to [23] emulsified with Freund complete adjuvant. Rabbits were immunized with the peptide antigen and three boosts were given at Days 7, 21, and 35. The antiserum was collected at Day 50 and was used for localization of fSP13 on fox spermatozoa using indirect immunofluorescence.

Fresh fox spermatozoa were washed and smears were prepared on glass slides, air dried, and fixed for 5 min with cold methanol. Slides were then covered with PBS containing 1% BSA for 45 min to block nonspecific antibody binding. They were then incubated at room temperature in a humidified chamber for 2 h with the rabbit sera diluted to 1:200 or with a control rabbit sera (Day 0) (1:200). Slides were then washed three times in PBS and a 1/500 dilution of goat anti-rabbit IgG fluorescein isothiocyanate-conjugated antibody (Sigma) diluted to 1:100 was added for 1 h. After three washings, slides were mounted with PBS:glycerol (1:1 v/v) and observed on an epifluorescent microscope (Olympus Optical Co., Hamburg, Germany). The pictures were obtained with the DP50 digital camera (Olympus).

Northern Blot Analysis

Northern blot hybridization was performed with a commercial kit (DIG Northern Kit, Roche Molecular, France) according to the manufacturer's instructions. Total RNA was isolated from specific tissues by using Trizol. Messenger RNAs were purified with Oligotex kit (Qiagen); concentration and purity were determined spectrophotometrically. One hundred nanograms of mRNA were separated on a 2% agarose gel and electrotransferred onto a Hybond-N+ membrane (Amersham Bioscience) and cross-linked to the membrane by ultraviolet irradiation. After blocking in the prehybridization buffer, the membrane was probed overnight at 68°C with the DIG-labeled fSP13 probe. Then the membrane was washed twice at 65°C with 2x SSC, 0.1% SDS, rinsed with washing buffer containing 0.1 M maleic acid, 0.15 M NaCl, and 0.3% Tween 20, and finally incubated for 30 min with anti-digoxygenin conjugated alkaline phosphatase diluted 1:10 000. Specific complexes were revealed by chemiluminescence detection according to the manufacturer's instructions (Roche Diagnostics). A control with a ß-actin probe was performed on the same membrane after dihybridization of the fSP13 probe.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of fSP13 as Auto- and Iso-Antigen

The localization of the fSP13 protein from the 2D gel is indicated by a white circle in Figure 1A. Immunoblotting of fSP13 purified by 2D gel electrophoresis with sera from rabbits and foxes immunized against fox sperm proteins [11] and vasectomized foxes [12] revealed strong immunoreactivity to a protein with a molecular size of 97 kDa and with several bands corresponding to partially degraded forms of fSP13 (Fig. 1B). This result confirms that fSP13 is the antigen localized on 2D gel in previous studies [11, 12], so this spot was used for further work, including Edman chemistry and mass spectrometry.



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  		FIG. 1. A) Silver-stained 2D gel of fox sperm proteins separated by isoelectric focusing (IEF) in the first dimension followed by SDS-PAGE in the second dimension (12% acrylamide). Isoelectric points (IP) are shown horizontally across the top and molecular masses are shown vertically on the left (kDa). Localization of the fSP13 spot is indicated with a white circle. B) Western blot analyses of purified fSP13 protein with antiserum from rabbits (iR1 and iR2) and foxes (iF2, iF4) immunized against fox sperm proteins (Verdier et al. [12]) and from vasectomized foxes vF1, vF2, vF3 (Verdier et al. [11]). fSP13 and degraded fragments of the protein are recognized by the different sera

Microsequencing of fSP13

To obtain structural information on the identity of fSP13, microsequencing of 20 spots of this protein was undertaken. A peptide sequence of 14 amino acids (AA) was obtained (Table 1). Database search using sequence information identified 92% of identity with the human proteins Fibrousheathin 2 (AF088868) and calcium-binding protein 86 (AY007205).


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  		TABLE 1. NH2-terminal sequence of fSP13 determined by Edman chemistry and homologies with proteins fibrousheathin 2 and calcium-binding protein 86

Cloning and Sequence Analysis of fSP13

To isolate the cDNA encoding fSP13, a primer pair designated from the sequence of the FSP2 homologue was used to amplify a 369-bp piece of cDNA by reverse transcription (RT)-PCR from testicular RNA. This amplified DNA probe was used to screen by Southern blot a cDNA library from fox testis. The nucleotide sequence of the full-length cDNA (AJ421969) consisted of 1662 bp, with an in-frame start codon at nucleotides 88–90 (Fig. 2). The cDNA contained a 1493-bp open reading frame (ORF) with untranslated regions of 87 bp at the 5' end and of 81 bp at the 3' end. The ORF encoded a protein of 498 AA with a predicted molecular weight of 53.1 kDa and an i.p. of 4.0. The peptide obtained by microsequencing the protein spot was recovered in the predicted AA sequence of the molecule (Fig. 2, black band).



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  		FIG. 2. Nucleotide and deduced amino acid sequences of the fox sperm protein fSP13. The numbers on the left refer to the nucleotide sequence, and the numbers on the right refer to the deduced amino acid sequence, which is shown below the cDNA sequence. The consensus ATG of the ORF and the termination codon (TGA) are indicated in bold letters. The black highlighted sequence indicates the position of the peptide obtained by microsequencing, and the gray highlighted band indicates the peptide sequence used for rabbit immunization. The nucleic acid sequence was submitted to the GenBank (Accession no. AJ421969)

Analysis of the predicted AA sequence revealed no apparent transmembrane regions. Comparison of the protein sequence with the Prosite database demonstrated the presence of 4 potential N-linked glycosylation sites (AA 32, 51, 118, 186), 10 potential casein kinase II phosphorylation sites (AA 16, 52, 75, 98, 104, 106, 311, 330, 423, and 472), 5 myristoylation sites (AA 20, 185, 398, 424, and 430), and 9 possible protein kinase C phosphorylation sites (AA 3, 145, 150, 330, 350, 385, 399, 405, and 456).

Comparison of the deduced fSP13 sequence to the GenBank data base using BLAST revealed that the fox sperm fSP13 had the closest AA similarity to two human sperm proteins (Fig. 3): fibrousheathin 2 (AF0088868, identity 60%, positive 69%) and testis-specific Calcium Binding Protein 86-VII (AF329634, identity 57%, positive 66%). The deduced 63 N-terminal AA sequence has similarities with the RII{alpha} domain that is also present in the sperm autoantigenic protein Sp17 (2842740; Fig. 4).



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  		FIG. 3. Homology comparison of the deduced amino acid sequence of fSP13 with those of the human sperm protein fibrousheathin 2 (AF329634) and testis-specific calcium binding protein 86-VII (AF329634). The sequences are listed in descending order of homology from fSP13. The AA sequences conserved by the three proteins are highlighted in black, and the AA sequences shared by two proteins are highlighted in gray



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  		FIG. 4. Alignments of fSP13, Sp17 (AF334735), and a consensus sequence of the N-terminal RII{alpha} binding domain. Highly conserved residues are boxed and highlighted in black; conservative substitutions are boxed and highlighted in gray

Mass Spectrometry

To confirm that the protein originally identified and cored from the gel was cloned and to validate the ORF deducted from the cDNA sequence, AA sequencing was performed by tandem mass spectrometry on peptides generated by overnight trypsin digestion at 37°C of the protein spot within pieces of the gel. The extracted peptides were concentrated and analyzed by MALDI-TOF mass spectrometry. Thirteen generated peaks of strong intensity were close to the theoretical results, calculated from the deducted AA sequence (Table 2).


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  		TABLE 2. Isotopic values (m/z) of peptides obtained of the fSP13 protein determined by MALDI-TOF mass spectrometry.a

Immunolocalization of fSP13 on Spermatozoa

Indirect immunofluorescence analysis of methanol-permeabilized fox spermatozoa using the rabbit serum localized fSP13 only to the principal piece of the majority of fox sperm (Fig. 5). Naive sera showed no immunofluorescence.



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  		FIG. 5. Indirect immunofluorescence localization of fSP13 on methanol-permeabilized fox spermatozoa. A) Contrast phase picture, (B) indirect-immunofluorescence micrography picture. The fSP13 protein is detected in the principal piece region of the flagellum where the fibrous sheath is localized

Analysis of fSP13 Expression on Fox Tissues

To localize where the gene coding for fSP13 is expressed in the fox, Northern blot was used with poly-A-enriched RNA isolated from various tissues. Messenger RNA coding for fSP13 is expressed at high levels in the testis (Fig. 6A), the band corresponds to a size of 1.6 kilobases. The fSP13 mRNA was not expressed in any other type of tissue, including brain, heart, kidney, spleen, lung, duodenum, epididymis, muscle, liver, ovary, vagina, and uterus. All the tissues tested were positive using ß-actin probe on the same membrane (Fig. 6B).



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  		FIG. 6. Analysis of fSP13 mRNA expression using Northern blot with mRNAs extracted from different fox tissues. fSP13 is specifically expressed in testis (A) whereas ß-actin used as quality control is detected in all tissues (B)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study aimed to characterize the antigenic protein fSP13 previously identified on fox spermatozoa by using different immunological methods, i.e., immunization with spermatozoa proteins or vasectomy [11, 12]. The auto- and iso-antigenicity of this sperm protein, synthesized in the testis, was here confirmed by Western blotting with the sera from the previous studies.

The cloning and sequence analysis of fSP13 was done using the methods Edman chemistry, RT-PCR, cDNA library, and mass spectrometry. The peptide sequence obtained by analysis of the 14 AA of the NH2 terminal sequence showed a 100% homology with FSP2. A primer pair designated from the terminal 14 AA and from another part of the sequence of FSP2 was used to amplify, by RT-PCR with testicular RNA, a band of 369 bp. This amplified cDNA was used as a probe to screen by Southern blot a cDNA library from fox testis. A sequence of 1662 bp was obtained, including a major ORF coding for 498 AA. The internal position of the 14 AA peptide sequence deduced from Edman chemistry could be explained by a preferential hydrolysis position on the fSP13 protein. During the purification or during the transfer on the PVDF membrane, the fSP13 protein has probably been cut. The ORF, in particular the presence of the AA situated in the N-terminal extremity (before the sequence determined by Edman sequencing), was confirmed by comparing the spectra of the digested protein and the theoretical peak values, deduced from the fSP13 sequence [24]. Moreover, these analyses showed the presence of various posttranscriptional modifications such as methylation. These data should be considered for further experiments including validation of a recombinant protein.

The apparent MW of fSP13 is 97 kDa, whereas the predicted MW is 53 kDa. Comparable differences between the predicted and apparent MW have been also described for different calcium-binding tyrosine phosphorylation-regulated proteins (CABYR), a homolog of fSP13 (respectively {approx}54 kDa and {approx}80 kDa for the mouse and {approx}53 kDa and {approx}86 kDa for the human). These differences have been explained by acidic i.p., which is also the case of fSP13 (4.3– 4.6) [25, 26]. Sequence analysis showed the existence of several glycosylation and phosphorylation sites, which suggests that posttranslational modifications may also be at the origin of the observed difference.

The fSP13 protein was shown to be testis specific. Northern blot analysis showed the expression of fSP13 mRNA only in the testis, but not in the numerous others tested tissues (brain, heart, kidney, spleen, lung, duodenum, epididymis, muscle, liver, ovary, vagina, uterus). This could explain the high antigenicity of the protein that is isolated from the immune system since puberty by the hematotesticular barrier.

The full-length cDNA of fSP13 presents homology with various sperm proteins, including FSP2, CABYR, and the RII{alpha} domain. The fox sperm fSP13 has the closest AA similarity (60%) to FSP2. Similarity of fSP13 and FSP2 are also apparent in their theoretical MW (53.1 and 53 kDa, respectively), their length (493 and 498 AA, respectively). The localization of fSP13 on the principal piece of the flagellum as indicated by indirect immunofluorescence is identical to the localization of FSP2.

The homology of fSP13 with FSP2 and mouse CABYR suggests that fSP13 is associated to the fibrous sheath, which might contribute to explaining its antigenicity. FSP2 is a human testis-specific protein associated with the sperm fibrous sheath [26]. The fibrous sheath has been described as an insoluble keratin-like structure with extensive disulfide linkage and has a modified form of the intermediate filament proteins in morphological, biochemical, physical, and immunological features [27]. Biochemical composition of the fibrous sheath is different in each species, and this work described for the first time a protein presumably associated with the fibrous sheath of the fox spermatozoa. Many fibrous sheath proteins have been identified as antigens, which can be explained by several arguments. First, as proteins from the outer dense fibers [28], they are unique to spermatozoa and are not produced until puberty, and then only in the developing spermatids in the testis. Thus, they may escape identification as self-protein during the development of the immune system and therefore stimulate an immune response when no longer sequestered behind the blood-testis and blood-epididymal barriers, e.g., after experimental immunization or vasectomy [11, 12]. Moreover, fibrous sheath proteins have a high content of disulfide bonds [29], protecting them from solubilization and degradation. Thus, they may represent a large supply of antigen that is released slowly over a long period of time and stimulates the immune system.

The significant homology of the C-terminal extremity of fSP13 with various calcium binding proteins (CBP), including the human CBP 86-VII, suggests that fSP13 may have calcium-binding properties. The CBPs of spermatozoa are interesting because the fertilizing capacity of spermatozoa is known to be largely dependent on Ca2+ signaling [30]. Several CBPs have been described in the spermatozoa, such as calmoduline; calmegin, a gamete-interacting protein; p50, an antigen involved in the acrosomic reaction; and CABYR [26, 31–33]. CABYR is a polymorphic, acidic (i.p. 4.0), testis-specific protein, which gains calcium-binding capacity when tyrosine-phosphorylated during in vitro capacitation [26]. It should be noted that fSP13, as CABYR, presents many phosphorylation sites. The similarities between CABYR and fSP13 are apparent in their AA sequence, their acidic properties, their localization on the principal piece of the sperm flagellum, and their testicular synthesis. It should be noted that the specific characteristics of the electrophoretic mobility of most CBPs on SDS-PAGE remain unexplained [30]. This could contribute to explaining why the MW of fSP13 determined on SDS-PAGE is different from those predicted from the cDNA sequence.

Properties of various fSP13 homologs (FSP2, CABYR, Sp17) also suggest an A-Kinase Anchoring Protein (AKAP)-binding activity for fSP13. The N-terminal extremity of fSP13 that is extremely similar to a sequence of FSP2, CABYR, and SP17 contains a motif similar to the protein kinase A regulatory subunit II{alpha} (RII{alpha}) [34, 35]. The N-terminus of the protein kinase A RII{alpha} contains a dimerization domain involved in the binding of AKAPs. Binding of AKAPs to proteins other than of the protein kinase A RII{alpha} has been described only for a few sperm-specific proteins including ropporin [36], the AKAP-associated sperm protein [37], CABYR [26], and Sp17 [34]. Like fSP13, all these molecules are colocalized with the sperm principal piece, including Sp17, that is mainly located within the sperm head [38] but has been recently shown to colocalize with AKAP3 along the length of the principal piece of the flagella [35]. Sp17 is a cytoplasmic protein in acrosome-intact spermatozoa, however, after the acrosome reaction Sp17 binds to the zona pellucida. The modification of localization of Sp17 is associated with proteolytic processing, resulting in the loss of its C-terminal calmoduline-binding site [39].

Concerning the potential functional activities of fSP13, the presence of a protein kinase A RII{alpha} homologous domain, the localization on the principal piece of the fox sperm and the functional activities of both human and mouse [25, 26] homologs of fSP13 suggest that this protein may be involved in sperm motility, e.g., in strengthening the cytoskeletal framework of the fibrous sheath to resist the mechanical forces of microtubule sliding [26].

Identification of sperm antigens can lead to the development of antisperm vaccines, which are attractive to control fertility in wild species. These vaccines have been shown to be active both in males and females (for a review, see [40]). For this use, the accessibility of the antibodies to the antigen is a critical point; therefore, good sperm antigens should have an extracellular domain or, at least, be exposed during the acrosome reaction. It should be noted that the prediction of protein localization from the protein sequence does not have an infallible predictive value to assess the contraceptive potential of an antigen. For instance, the Sp17 protein sequence does not contain any transmembrane domain [41] however, this antigen has been used for the development of a contraceptive vaccine [42]. The use of fSP13 as a target for a contraceptive vaccine should now be assessed.

The CABYR murine homolog of fSP13 has been suggested to be used as a target for contraceptive drug development in the murine model [25]. The identification of the iso- and auto-antigenic fSP13 fox sperm protein similar to mouse and human sperm CABYR provides a unique opportunity for the development of an immunocontraceptive or other contraceptive drug in canid species.


   ACKNOWLEDGMENTS
 
The authors are grateful to Pr. Botond Penke for helpful discussions, to Franck Saulnier and Dr. Gerard Humbert for the excellent sequencing of the protein, and to Dr. Nathalie Monhoven for kindly sequencing DNA. We thank the Entente Interdepartementale de Lutte contre la Rage and the Oral Vaccination Unit of the AFSSA Nancy for providing wild foxes, and Michel Munier and Jacqueline Bailly for excellent technical assistance.


   FOOTNOTES
 
1 Supported by the research grant 625 11 N 2042 EPR 98-02 from the DGAl of the French Ministry of Agriculture. Back

2 Correspondence: F. Boué, AFSSA Nancy, Domaine de Pixérécourt, BP 9, F-54220 Malzéville, France. FAX: 33 3 83 29 89 59; f.boue{at}nancy.afssa.fr Back

Received: 2 June 2004.

First decision: 28 June 2004.

Accepted: 30 September 2004.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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